Eliminating malignant contamination from therapeutic human spermatogonial stem cells
Eliminating malignant contamination from therapeutic human spermatogonial stem cells
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DOI：
10.1172/jci65822
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发表时间：
2013-04-01
影响因子：
15.9
通讯作者：
Orwig, Kyle E.
中科院分区：
文献类型：
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作者：
Dovey, Serena L.;Valli, Hanna;Orwig, Kyle E.
精原干细胞（SSC）移植已被证明可以恢复多个物种的生育能力，并可用于治疗某些男性不育症（例如继发于癌症性腺毒性治疗的不育症）。为了确保这种生育力保存策略的安全性，需要从人类睾丸细胞悬液中分离和富集 SSC 并去除恶性污染的方法。我们使用流式细胞术来表征人睾丸细胞和白血病细胞（MOLT-4 和 TF-1a）上的细胞表面抗原表达。我们通过 FACS 证明 EpCAM 由人类精原细胞表达，但 MOLT-4 细胞不表达。相比之下，HLA-ABC 和 CD49e 标记 > 95% 的 MOLT-4 细胞，但在人精原细胞上不表达。对 MOLT-4 污染的人睾丸细胞悬液进行多参数分类，以分离 EpCAM(+)/HLA-ABC(-)/CD49e(-)（假定的精原细胞）和 EpCAM(-)/HLA-ABC(+)/CD49e(+)（假定的 MOLT-4）细胞组分。 EpCAM(+)/HLA-ABC(-)/CD49e(-) 组分富集精原细胞定植活性，并且在人对裸鼠异种移植后不会形成肿瘤。 EpCAM(-)/HLA-ABC(+)/CD49e(+) 部分在异种移植后产生肿瘤。这种方法可以通过稍加修改来推广，以去除污染的 TF-1a 白血病细胞。因此，FACS提供了一种通过利用细胞表面抗原表达的差异来分离和富集人类精原细胞并去除恶性污染的方法。
Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4-contaminated human testicular cell suspensions was performed to isolate EpCAM(+)/HLA-ABC(-)/CD49e(-) (putative spermatogonia) and EpCAM(-)/HLA-ABC(+)/CD49e(+) (putative MOLT-4) cell fractions. The EpCAM(+)/HLA-ABC(-)/CD49e(-) fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to-nude mouse xenotransplantation. The EpCAM(-)/HLA-ABC(+)/CD49e(+) fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.